PROJECT SUMMARY/ABSTRACT The reprogramming of abundant and accessible cells into therapeutically useful cell types holds great promise for regenerative medicine. Cellular reprogramming can be achieved by ectopically expressing transcription factors (TFs) that directly convert one differentiated cell type into another, bypassing embryonic states in an attempt to boost the speed and efficiency of target cell production. A number of different cell types have been generated by such ?direct lineage reprogramming? methods, but their practical utility has been limited because, in most protocols, only a small percentage of cells are successfully converted to the target cell type. Even then, the resulting populations are often partially differentiated or incompletely specified. Most cell engineering methods require the use of at least one pioneer factor, a unique class of TFs that are able to access their binding sites in silent chromatin. Pioneer TFs have the capacity to impart lineage competence in a context- specific manner, and play central roles in development, as reflected by their redeployment across disparate developmental programs. Our long-term goal is to understand the mechanism of pioneer factor-mediated direct lineage reprogramming. In this proposal we employ prototypical pioneer TFs, the FoxA family, to drive conversion of fibroblasts to an endoderm progenitor-like (iEP) state, representing a paradigm for direct lineage reprogramming. Based on our preliminary results and current evidence, we hypothesize that during direct lineage reprogramming, pioneer TFs re-engage developmental GRNs, depending on the chromatin state of the cells into which they are introduced. Our three independent yet related aims are directed at understanding: (Aim 1) the nature of transcriptional changes during reprogramming from their origin and their relation to developmental programs; (Aim 2) the direct targets of FoxA pioneer TFs and their cofactor, Hnf4a and their activity to drive fate change; (Aim 3) the influence of chromatin context on target accessibility of pioneer TFs and how this impacts efficiency of reprogramming and the potential of cells generated. Here we apply an innovative single-cell lineage tracing methodology and genomic technology to record TF binding history in cells undergoing reprogramming. Together, this will generate an unprecedented digital quantification of the reprogramming process, and will reveal barriers to the successful conversion of cell identity. An improved understanding of pioneer-mediated reprogramming mechanisms will facilitate enhanced conversion efficiency and fidelity across an array of reprogramming strategies, and improve knowledge of the action of this important class of transcriptional regulators.